Previous work in the LCS has shown that opioid peptides can directly modulate cardiac myocyte contractile function and inhibit catecholamine induced inotropy by affecting signal transduction through the beta- adrenergic pathway. Since both contractile force and beta-adrenergic mediated events are involved in the hypertrophic process, we sought to determine whether the cardiac response to hypertrophic stress is associated with changes in opioid peptide levels in rat heart, whether opioid peptide production is regulated at the level of gene expression, and investigated factors that regulate opioid gene expression in the heart. We studied opioid peptide production and/or proenkephalin (PNK) gene expression in 3 well established models of cardiac hypertrophy: aging, aortic constriction and alpha-adrenergic stimulation in culture. Cardiac opioid peptide was upregulated 2-3-fold with advancing age, and PNK gene expression was elevated in the heart during aging (7-fold), and during chronic pressure overload hypertrophy leading to heart failure (2-fold). Paradoxically, PNK gene expression was transiently downregulated 3 days after aortic constriction, and this transient decrease was abolished by prior chemical sympathectomy. Norepinephrine (NE) treatment increased the level of PNK mRNA in myocyte cultures (peak 4 hr). Neither alpha- nor beta-adrenergic receptor stimulation alone was sufficient to produce PNK induction. The induction of PNK by NE in myocytes did not occur unless myocytes were co-cultured with cardiac fibroblasts (in situ hybridization), and could not be reproduced with conditioned media from cardiac fibroblasts. The induction of hypertrophy marker genes (ANF and alpha-skeletal actin) by NE in myocyte cultures was not diminished by adding opioid peptides to the media, nor augmented by supplementing the media with opioid receptor antagonists. In summary, opioid peptide production is augmented during chronic cardiac hypertrophy, and is regulated, in part, at the level of gene expression. The negative modulatory influence of opioid peptides on cardiac myocytes may be limited to functional parameters, and apparently does may not affect hypertrophic growth of myocytes.